Journal: Frontiers in Cell and Developmental Biology

Article Title: Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis

doi: 10.3389/fcell.2021.672009

Figure Lengend Snippet: A subset of tBHP-RBCs localized to early and late endosomes. bEnd.3 cells were stained for early endosome antigen 1 (EEA-1; red) (A) , lysosome–associated membrane protein 1 (LAMP-1; red) (B) , and 4′,6-diamidino-2-phenylindole (DAPI; blue) after a 48-h incubation with PBS- or tBHP-RBCs. RBCs are indicated by yellow arrowheads in panels (A,B) . Massive accumulation of tBHP-RBCs within bEnd.3 cells is shown in panel (B) . Orthogonal projections from z-stacks of confocal images show tBHP-RBCs co-localized with the endosomal markers in bEnd.3 cells (white arrowheads in A,B ). Scale bar = 20 μm. Quantification of RBC attachment (expressed as % of number of bEnd.3 cells), RBCs localized to the nucleus (expressed as % of total RBCs; also see Supplementary Figure 2 ) , EEA-1- and LAMP-1-positive RBCs (expressed as % of total RBCs), and RBCs in the nucleus positive for EEA-1-/LAMP-1 (expressed as a % of RBCs in the nucleus) (EEA-1: C and LAMP-1: D ). All experiments were repeated at least three times.

Article Snippet: Cells were incubated with Alexa Fluor 647-labeled anti-early endosome antigen 1 (EEA-1) antibody (1:50 in PBS with 0.75% bovine serum albumin and 0.1% Triton X-100; Santa Cruz Biotechnology, Dallas, TX, United States) or rat anti-lysosome-associated membrane protein 1 (LAMP-1) antibody (the original antibody was diluted 1:2 in 50% glycerol followed by 1:5 dilution in PBS with 0.75% bovine serum albumin and 0.1% Triton X-100; Developmental Studies Hybridoma Bank, Iowa City, IA, United States) overnight at 4°C.

Techniques: Staining, Membrane, Incubation

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1 signaling regulates ER calcium levels. Schematic (I) Timeline of experiments assessing changes in ER calcium levels. Schematic (II) Timeline of experiments, in cells treated with siRNA before assessing changes in ER calcium levels. (A) Fluo4 fluorescence in HEK cells expressing NIC1-RFP ( ) or RFP ( ), at time-t (F t ) relative to onset (F 0 ) measured in calcium free medium at baseline and in response to 2 μM TG. (B) YFP/CFP ratio in HEK cells co-transfected with D1ER and NIC1-RFP ( ) or, Bcl-xL-RFP ( ) or, RFP ( ) and cultured for 36 h before imaging. (C) Schematic (not to scale) depicting - IP3R3, Grp75, VDAC1 and MCU- in the context of ER and mitochondria. (D–F) Ratio of YFP/CFP fluorescence in HEK cells, imaged 36 h after co-transfection with D1ER and NIC1-RFP ( ) or RFP ( ), in cells pre-treated with siRNA to IP3R3 (D-ii) or VDAC1 (E-ii) or MCU (F-ii) or scrambled control ( D-F-i , NIC1-RFP ( ) and RFP ( ). (D–F iii) Percent mRNA levels of the genes as shown in panels, in cells treated with siRNA to IP3R3 ( D , N=3) or VDAC1 ( E , N=2) or MCU ( F , N=3) and scrambled control. Data plotted as mean ± SD of the indicated number of cells in 6-12 fields across three (A, D, F) or two (B, D) independent experiments. *** indicates significant difference at all time points with p-values ≤0.001 and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Fluorescence, Expressing, Transfection, Cell Culture, Imaging, Cotransfection, Control

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1-mediated Treg survival requires IP3Rs, Grp75 and MCU. (A) Percent apoptotic nuclei in activated WT Tregs cultured without IL-2 for 24 h with vehicle control (UT) or, 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (B, C) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or NIC1-NES (B) or pMIG or Bcl-xL (C), and cultured without IL-2 for 24 h with vehicle control, or 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (D) Basal and maximum OCR, computed as described in methods, in Notch1 -/- Tregs transduced with pBABE or NIC1-NES. (E) Immunoblot of whole cell lysates prepared from activated WT Tregs cells cultured with or without 10 μM GSI-X or 10 μM MKT-077 for 8 h and the samples run in duplicate. Membrane was probed either for (p)PDH, Notch1 and Tubulin (E-i) or PDH, Notch1 and Tubulin (E-ii) . The immunoblot is representative of two independent experiments. Data are mean ± SD of three independent experiments (A–C) and readings in 4 wells from two independent experiments (D). ** indicates significant difference with p-value ≤0.01 and ns, not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Cell Culture, Control, Transduction, Western Blot, Membrane

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Notch1 activity regulates Grp75 protein levels. (A) Immunoblots of whole cell lysates prepared from activated Notch1 +/+ (Cre-ve) and Notch1 -/- (Cre+ve; Cd4-Cre::Notch1 lox/lox ) Tregs, run in duplicate. Membranes were sequentially probed for Grp75, VADC1 (*VDAC1 band), and Actin (A-i) or Notch1, MCU and Actin (A-ii). Mean ± SD values below are the densitometry analysis of Grp75, VDAC1 and MCU relative to Actin. (B) Representative Z-projected confocal images of Notch1 +/+ and Notch1 -/- Tregs immune-stained with an antibody to Grp75 (green). scale bar: 5 μm. Images are representative of 106 Notch1 +/+ Tregs and 82 Notch1 -/- Tregs. (C) Relative transcript levels of indicated genes in Notch1 +/+ and Notch1 -/- activated Tregs. (D) Immunoblots of cell lysates from Notch1 -/- Tregs transduced with pBABE or NIC1-NES probed for Notch1, Grp75 and Actin. Mean ± SD values below are the densitometry analysis of Grp75 relative to Actin. (E) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or Grp75, cultured without IL-2 for 24 h with 5μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (F) Basal and maximum OCR in Notch1 -/- Tregs transduced with pBABE or Grp75. Control (pBABE) condition in panels E and F are common Figures 5B, D as these were tested in the same experiment, as described in methods . (G, H) Cell lysates of WT activated Tregs cultured for 6 h without IL-2 were subject to immunoprecipitation using an antibody to Notch1 (G) or Grp75 (H) or, IgG (Isotype control), and associated proteins analyzed by western blotting for Grp75, VDAC1 (*shows VDAC1), Notch1, Vps34, Actin and IgG (Isotype control). Immunoblots are representative of three independent experiments (A) or, two independent experiments (D, G, H) . Data show the mean ± SD of three independent experiments (C, E) and readings in 4 wells from two independent experiments (F). * and ** indicates significant difference with p-value ≤0.05 and ≤0.01 respectively, and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Activity Assay, Western Blot, Staining, Transduction, Cell Culture, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Summary of key outcomes. Non-nuclear localized NIC1 interacts with Grp75 and VDAC1 in a complex that modulates calcium homeostasis in mitochondria with consequences to cellular metabolism and survival. The schematic is not to scale.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques:

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Structural features of and interaction model for Grp94 and myocilin. ( A ) Model of Grp94 highlighting its structural domains: Pre-N subdomain, cyan; N-terminal ATP-binding domain (N), light blue; middle domain (M), blue; C-terminal dimerization domain (C), purple. The second monomer of the obligate homodimer is shaded gray for clarity. ( B ) Schematic of myocilin structural organization emphasizing the C-terminal olfactomedin (OLF) domain in green. Colored spheres represent calcium (orange) and potassium (magenta) ions. Models are not drawn to scale. Models are from structures with PDB-ID codes 5ULS (Grp94) and 4WXQ, 5VR2 (myocilin). ( C ) Model of Grp94 involvement in myocilin aggregation and glaucoma-associated cellular toxicity. Grp94 is recruited by misfolded, aggregating mutant myocilin via the ERAD system, but counterproductively facilitates aggregation and preserves toxic aggregates; preventing the Grp94/myocilin interaction is a viable method of rescuing cells from cytotoxicity via alternative clearance mechanisms. Myoc = myocilin.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Binding Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: The N-terminal domain of Grp94 is responsible for the aberrant Grp94/OLF protein-protein interaction. ( A ) Grp94 N enhances the rate of OLF aggregation, whereas Grp94 MC appears to stabilize OLF against aggregation as indicated by ThT fluorescence. Results represent the average of 20 (OLF + Grp94 N ), 12 (OLF + Grp94 NM ), and 12 (OLF + Grp94 MC ) replicates from at least 2 biological replicates. The ^ symbol represents data presented previously ; ***(p < 0.0001) represents statistically significant differences relative to OLF at 24 hours. ( B ) The Grp94 N-terminal domain and OLF co-aggregate over the course of the aggregation assay in ( A ). S = supernatant, W = wash, and P = pellet/aggregate. See Fig. for full four-day kinetics assay data, and Fig. for additional co-aggregation SDS-PAGE gels for the remaining domain constructs.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Fluorescence, SDS Page, Construct

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Impact of Grp94 domains on OLF aggregation.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques:

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Mapping the Grp94/OLF protein-protein interaction interface. ( A ) Coomassie blue (left) and dansyl fluorescence (right) visualization of crosslinking reaction products (arrows) as seen by SDS-PAGE for the coupling of Grp94 NM with OLF. Dashed line distinguishes different visualizations of same gel. See Fig. for complementary reactions with Grp94 N and Grp94 MC . See Fig. for uncropped gel images. ( B ) MS spectrum of crosslinked peptides containing the Grp94 K547 -OLF K468 linkage (top). HCD fragmentation spectrum and fragment mass match accuracy (1 of 9 PSMs observed) with y- and b-ion assignments (inset) consistent with the crosslinked peptides shown (bottom). Data correspond to the sample gel shown in Fig. ; refer to Table for additional details. ( C ) Map of the interaction interfaces between Grp94 and OLF identified by mass spectrometry. Protein domains demarcated by first amino acid residue in the domain (top, Grp94; bottom, OLF). Colors match those of the models in Fig. .

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Fluorescence, SDS Page, Mass Spectrometry, Residue

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Model of Grp94 protein-protein interaction sites with OLF. ( A ) Top: structural model of Grp94 highlighting both the aberrant (K72) and stabilizing (K547) interaction sites detected by crosslinking/MS (see Table ). Bottom: electrostatic surface potential view of boxed MC region; Grp94 MC model PDB-ID: 2O1T, which contains 18 additional, largely acidic C-terminal residues compared to 5ULS shown above (also see Fig. ). The proposed OLF recognition surface is identified by dotted circle. ( B ) Top: top-down perspective of OLF β-propeller, with unstructured N-terminal aggregation-associated Grp94 recognition site marked as Nterm*. Middle: side-on view of OLF with stabilizing interaction residue (K468) featured. Bottom: electrostatic surface potential of OLF in same pose as middle panel, indicating a positively-charged surface. The surface potential is colored negative (red, −5 kT/e − ) to positive (blue, 5 kT/e − ). The remaining color scheme and models shown are the same as in (Fig. ) unless otherwise specified.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Residue

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: The Pre-N region and conformational state of Grp94 N are critical for the aberrant interaction with myocilin OLF. ( A ) Truncation of the N domain of Grp94 eliminates its capacity to enhance the rate of OLF aggregation; residues 22-57 appear crucial to the interaction. Results represent the average of 12 replicates (for both OLF + Grp94 58-804 and OLF + Grp94 73-804 ) from 2 biological replicates. The ^ symbol represents data previously published ; ***(p < 0.0001) represents statistically significant differences relative to OLF + Grp94 FL . ( B ) Despite diminished effects on OLF aggregation, the majority of the Grp94 truncation variant proteins still co-aggregate with OLF. S = supernatant, W = wash, P = pellet/aggregate. ( C ) Treatment of Grp94 N with Grp94-specific inhibitor 4-Br-BnIm mitigates its interaction with OLF. The traces for Grp94 N (for comparison) are the same as in Fig. ; results for OLF + Grp94 N + 4-Br-BnIm represent the average of 9 replicates from 2 biological replicates. ***(p < 0.0001) represents statistically significant differences relative to OLF + Grp94 N . D) Grp94 N is partially rescued from its co-aggregation fate with OLF in the presence of inhibitor 4-Br-BnIm. Abbreviations given in ( B ).

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Variant Assay, Comparison